Muc1-derived peptide, and pharmaceutical composition for treatment or prevention of cancer, immunity-inducing agent and method for manufacturing antigen presenting cell using same

ABSTRACT

The present invention provides a peptide that includes eight or more consecutive amino acid residues of amino acid sequence of one of Sequence ID Nos. 1 to 12 and that consists of eleven or less amino acid residues.

TECHNICAL FIELD

The present invention relates to an MUC1-derived peptide, morespecifically an immunogenic peptide that binds to a human leukocyteantigen and thus presents an antigen to a T cell, and a pharmaceuticalcomposition for treatment or prevention of cancer, an immunity-inducingagent, a method for manufacturing an antigen presenting cell and thelike, using the same.

BACKGROUND ART

It is thought that cancer cells always occur accidentally in livingbodies. It is likely that a reaction in which specific cancer antigensderived from cancer cells are eliminated by natural immunity usuallyoccurs, and then a specific immune response is induced, so that areaction in which the cancer cells are eliminated by lymphocytes and thelike occurs.

The formation of a complex between a human leukocyte antigen (HLA) thatis present on the cell surface and a lymphocyte is necessary forrecognition of antigens derived from cancer cells. HLA molecules, whichare major histocompatible antigens, are broadly divided into class-Imolecules (HLA-A, -B, and -C) and class-II molecules (HLA-DP, -DQ, and-DR). T cell antigen receptors (TCRs) on cytotoxic T cells (CTLs)specifically recognize cancer antigens (CTL epitopes) consisting of 8 to11 amino acids presented to HLA class-I molecules on the surfaces ofcancer cells, and thus a reaction in which cancer cells are eliminatedby the CTLs is induced.

Nowadays, a search for immunogenic peptides is made with the expectationof the application to treatment or prevention of various immunologicaldiseases. For example, JP H8-151396A discloses that oligopeptides havingspecific amino acid sequences have an HLA-binding property.

CITATION LIST Patent Document

Patent Document 1: JP H8-151396A

SUMMARY OF INVENTION Technical Problem

Although a large number of peptides having an HLA-binding property areknown, further peptides that can be used for treatment or prevention ofvarious cancers are required. The gene for HLA is rich in polymorphism,and therefore, multi-type immunogenic peptides that can be applied to aplurality of types of HLAs are also required.

Solution to Problem

The present invention has been achieved in light of the aforementionedcircumstances, and an object thereof is to provide an immunogenicpeptide that binds to an HLA class-I molecule, particularly a peptidethat can induce CTLs, and a pharmaceutical composition for treatment orprevention of cancer, an immunity-inducing agent, a method formanufacturing an antigen presenting cell and the like, using the same.

That is, the present invention encompasses the following aspects of theinvention.

(1) A peptide including eight or more consecutive amino acid residues ofamino acid sequence of one of Sequence ID Nos. 1 to 12, and consistingof eleven or less amino acid residues.

(2) The peptide according to aspect (1), wherein one or several aminoacids in the amino acid sequence are substituted, inserted, deleted, oradded, and the peptide has immunogenicity.

(3) The peptide according to aspect (2), wherein an amino acid at a2-position of the amino acid sequence is substituted with tyrosine,phenylalanine, methionine, tryptophan, valine, leucine or glutamine,and/or a C-terminal amino acid is substituted with phenylalanine,leucine, isoleucine, tryptophan, methionine, or valine.

(4) A pharmaceutical composition for treatment or prevention of cancer,comprising the peptide according to any one of aspects (1) to (3).

(5) The pharmaceutical composition according to aspect (4), which is ina form of vaccine.

(6) The pharmaceutical composition according to aspect (4) or (5),wherein the peptide can bind to one or more types of HLA molecules.

(7) An immunity-inducing agent comprising the peptide according to anyone of aspects (1) to (3).

(8) The immunity-inducing agent according to aspect (7), which is usedto induce a cytotoxic T cell.

(9) The immunity-inducing agent according to aspect (7) or (8), whereinthe peptide can bind to one or more types of HLA molecules.

(10) A method for manufacturing an antigen presenting cell having aCTL-inducing activity, the method comprising a step of bringing thepeptide according to any one of aspects (1) to (3) into contact with anantigen presenting cell in vitro.

Advantageous Effects of Invention

In recent years, an immunotherapy has been attracting attention astreatment of cancer. The peptide of the present invention has a highHLA-binding property and a high ability to induce CTLs, and is thusstrongly expected to be useful as a cancer vaccine. Moreover, it islikely that the peptide of the present invention can be applied tovarious immunotherapies, particularly to a dendritic cell therapy.

Mucin is a large glycoprotein that is expressed in various epithelialcells including normal cells and malignant cells (Devine P L andMcKenzie I F: Mucins: structure, function, and associations withmalignancy. Bioessays 14: 619-625, 1992). MUC1, which is one of mucinpolypeptides, is a unique glycoprotein that is expressed passing throughthe cell membrane on a cell surface (Gendler S J, Lancaster C A,Taylor-Papadimitriou J, Duhig T, Peat N, Burchell J, Pemberton L, LalaniE N and Wilson D: Molecular cloning and expression of humantumor-associated polymorphic epithelial mucin. J Biol Chem 265:15286-15293, 1990).

MUC1 of cancer cells undergoes incomplete glycosylation (Hanisch F G,Stadie T R, Deutzmann F and Peter-Katalinic J: MUC1 glycoforms in breastcancer—cell line T47D as a model for carcinoma-associated alterations ofO-glycosylation. Eur J Biochem 236: 318-327, 1996), and it has beenreported that killer T cells against MUC1 are induced in pancreaticcancer, breast cancer, ovarian cancer, and the like (20 Barnd D L, Lan MS, Metzgar R S and Finn O J: Specific, major histocompatibilitycomplex-unrestricted recognition of tumorassociated mucins by humancytotoxic T cells. Proc Natl Acad Sci USA 86: 7159-7163, 1989.

21 Jerome K R, Barnd D L, Bendt K M, Boyer C M, Taylor-Papadimitriou J,McKenzie I F, Bast R C Jr and Finn O J: Cytotoxic T-lymphocytes derivedfrom patients with breast adenocarcinoma recognize an epitope present onthe protein core of a mucin molecule preferentially expressed bymalignant cells. Cancer Res 51: 2908-2916, 1991.

22 Ioannides C G, Fisk B, Fan D, Biddison W E, Wharton J T and O'Brian CA: Cytotoxic T cells isolated from ovarian malignant ascites recognize apeptide derived from the HER-2/neu protooncogene. Cell Immunol 151:225-234, 1993.).

Specific aspects of the peptide of the present invention can bind to aplurality of types of HLAs. Therefore, with the peptide of the presentinvention, a cancer vaccine, a dendritic cell therapy, and the like thatcover an extremely broad group of cancer patients can be provided.

BRIEF DESCRIPTION OF DRAWINGS

FIG. 1 shows results (the number of IFN-γ producing cells) of ELISAassay when samples from a patient (HLA type: 24:02/33:03) that hasundergone a dendritic cell/CTL therapy against MUC1 are stimulated usingpeptides of Sequence ID Nos. 1, 3, 5, and 10.

FIG. 2 shows results (the number of IFN-γ producing cells) of ELISAassay when samples from a patient (HLA type: 24:02/33:03) that hasundergone a dendritic cell/CTL therapy against MUC1 are stimulated usingpeptides of Sequence ID Nos. 1, 3, 5, and 10.

FIG. 3 shows results (the number of IFN-γ producing cells) of ELISAassay when samples from a patient (HLA type: 02:06/24:02) that hasundergone a dendritic cell/CTL therapy against MUC1 are stimulated usingpeptides of Sequence ID Nos. 1, 3, 5, and 10.

FIG. 4 shows results (the number of IFN-γ producing cells) of ELISAassay when samples from a patient (HLA type: 02:01/02:06) that hasundergone a dendritic cell/CTL therapy against MUC1 are stimulated usingpeptides of Sequence ID Nos. 1, 2, 3, 5, 10, and 11.

DESCRIPTION OF EMBODIMENTS

1. Immunogenic Peptide

A peptide according to the present invention is a peptide that includeseight or more consecutive amino acid residues of the amino acid sequenceof one of Sequence ID Nos. 1 to 12, and that consists of eleven or lessamino acids, preferably ten or less amino acids, and more preferablynine or less amino acids, in total. The peptide of the present inventionmay also have the amino acid sequence of one of Sequence ID Nos. 1 to12. The peptide of the present invention is derived from mucin 1 (MUC1),which is one of tumor-associated antigens.

Selected were the amino acid sequences that were based on the amino acidsequence of MUC1 and whose HLA molecule-binding properties predictedbased on the hypothesis obtained by using an active learning experimentmethod (JP H11-316754A) were 3 or more in terms of a −log Kd value.

Table 1 below shows the amino acid sequences for the peptide of thepresent invention, and the predicted HLA-binding scores.

TABLE 1 Amino acid sequence Predicted binding score (Sequence ID No.)Location in MUC1 to A*24:02 to A*02:01 to A*02:06FLGLSNIKF (Seq. ID No. 1) 1086 5.0955 4.4235 4.1939SVPVTRPAL (Seq. ID No. 2)  100 5.0767 4.4656 5.8224GVPGWGIAL (Seq. ID No. 3) 1155 4.9066 4.5398 5.3254AFREGTINV (Seq. ID No. 4) 1106 4.6737 4.6098 4.4007AASRYNLTI (Seq. ID No. 5) 1128 4.5613 4.2522 4.1161LQRDISEMF (Seq. ID No. 6) 1069 5.4895 3.9363 3.812HHSDTPTTL (Seq. ID No. 7)  997 5.2785 3.8534 4.0609SFFFLSFHI (Seq. ID No. 8) 1041 5.0343 4.5581 3.958TLAFREGTI (Seq. ID No. 9) 1104 4.8467 4.8911 3.9425STGVSFFFL (Seq. ID No. 10) 1037 4.7519 4.0289 5.2728GQDVTSVPV (Seq. ID No. 11)   95 3.7866 5.1026 6.1919FSAQSGAGV (Seq. ID No. 12) 1148 3.0319 5.2427 4.0462

The peptide of the present invention has an HLA-binding property andimmunogenicity (also referred to merely as “HLA peptide” or “immunogenicpeptide” hereinafter). “Immunogenicity” as used herein means an abilityto induce an immune reaction, and, for example, means “having aCTL-inducing activity and thus a cytotoxic activity against cancercells”.

In a preferred aspect, the peptide of the present invention is amulti-HLA peptide that can bind to a plurality of types of alleles ofthe HLA-A gene A. For example, the peptide of Sequence ID No. 10strongly binds to an HLA-A*24:02 gene product (HLA-A*24:02 molecule), anHLA-A*02:01 gene product (HLA-A*02:01 molecule), and an HLA-A*02:06 geneproduct (HLA-A*02:06 molecule), and has a high immunogenicity.

HLA subtypes to which the peptide of the present invention can bind arenot limited to the HLA-A*24:02, the HLA-A*02:01, and the HLA-A*02:06.However, about 85% of the Oriental including Japanese and about 55% ofthe Occidental have these HLA subtypes, and therefore, it is thoughtthat the multi-HLA peptide has a high patient cover ratio in animmunotherapy or the like.

Amino acid residues of the amino acid sequences of Sequence ID Nos. 1 to12 or portions thereof may be modified as long as the peptide of thepresent invention retains immunogenicity. The amino acid sequences ofSequence ID Nos. 1 to 12 are intended to be presented on antigenpresenting cells, but, when being administered directly to the body, thepeptide of the present invention may undergo a modification such asdigestion of its terminus in the digestive organs depending on theadministration route. Therefore, prior to being taken up by an antigenpresenting cell, the peptide of the present invention may exist in aprecursor form in which one or more amino acid residues and the like areadded to the N-terminus and/or C-terminus such that the peptide of thepresent invention retains the amino acid residues of Sequence ID Nos. 1to 12 when binding to a predetermined HLA class-I molecule on theantigen presenting cell.

Furthermore, in the peptide of the present invention, one or severalamino acid residues of the peptide of the present invention may besubstituted, inserted, deleted, or added, and/or the peptide of thepresent invention may undergo modifications such as glycosylation,side-chain oxidation and/or phosphorylation. The “amino acids” are usedherein in their broadest sense, and include artificial amino acidvariants and artificial amino acid derivatives in addition to naturalamino acids. In this specification, examples of the amino acids includenatural protein L-amino acids; D-amino acids; chemically modified aminoacids such as amino acid variants and amino acid derivatives;non-protein natural amino acids such as norleucine, β-alanine, andornithine; and chemically synthesized compounds having characteristicsknown in the art as features of amino acids. Examples of non-naturalamino acids include α-methylamino acids (e.g., α-methylalanine), D-aminoacids, histidine-like amino acids (e.g., β-hydroxy-histidine,homohistidine, α-fluoromethyl-histidine, and α-methyl-histidine), aminoacids with side chains including additional methylenes (i.e., “homo-”amino acids), and substituted amino acids (e.g., cysteic acid) in whicha carboxylic acid functional group in a side chain is substituted with asulfonic acid.

Regarding the substitution of amino acid residues and the like, a personskilled in the art could substitute the amino acids of the peptide ofthe present invention as appropriate in consideration of the regularityof peptide sequences exhibiting an HLA-binding property (J. Immunol.,152: p 3913, 1994; Immunogenetics, 41: p 178, 1995; J. Immunol., 155: p4307, 1994).

More specifically, in the case of a peptide that binds to theHLA-A*24:02 molecule, the amino acid at the 2-position of the peptidemay be substituted with tyrosine, phenylalanine, methionine, ortryptophan, and/or the C-terminal amino acid may be substituted withphenylalanine, leucine, isoleucine, tryptophan, or methionine. In thecase of a peptide that binds to the HLA-A*02:01 molecule, the amino acidat the 2-position may be substituted with leucine or methionine, and/orthe C-terminal amino acid may be substituted with valine or leucine.Furthermore, in the case of a peptide that binds to the HLA-A*02:06molecule, the amino acid at the 2-position may be substituted withvaline or glutamine, and/or the C-terminal amino acid may be substitutedwith valine or leucine.

All aspects of the peptide of the present invention can be manufacturedusing methods known to a person skilled in the art. For example, thepeptide of the present invention may be synthesized artificially using asolid phase method such as the Fmoc method or the tBoc method, or aliquid phase method. Moreover, a desired peptide may also bemanufactured by expressing a polynucleotide coding for the peptide ofthe present invention or a recombinant vector including thepolynucleotide. All of the thus obtained peptides can be identifiedusing a method known to a person skilled in the art. For example, thepeptides can be identified using the Edman degradation method, massspectrometry, or the like.

2. Pharmaceutical Composition

A pharmaceutical composition for treatment or prevention of canceraccording to the present invention contains, as an active component, apeptide that includes eight or more consecutive amino acid residues ofone or more amino acid sequences selected from the group consisting ofSequence ID Nos. 1 to 12, and that consists of eleven or less aminoacids, preferably ten or less amino acids, and more preferably nine orless amino acids, in total, for example. The peptide contained in thepharmaceutical composition may also have the amino acid sequence of oneof Sequence ID Nos. 1 to 12. The peptide is as defined above.

The peptide of the present invention is presented on an antigenpresenting cell and thus induces CTLs, and the induced CTLs injurecancer cells. Therefore, the active component of the pharmaceuticalcomposition of the present invention is not limited to the peptide ofthe present invention, and may be a component that can induce CTLsdirectly or indirectly. For example, a polynucleotide coding for thepeptide or a vector including the polynucleotide, an antigen presentingcell that presents a complex between the peptide and an HLA molecule onits surface, or an exosome secreted by the antigen presenting cell, orcombinations thereof may be used. Examples of the antigen presentingcell to be used include a macrophage and a dendritic cell, and it ispreferable to use a dendritic cell, which has a high ability to induceCTLs. The pharmaceutical composition of the present invention may alsocontain other components known to be used for treatment of cancer, suchas a chemokine, a cytokine, a tumor necrosis factor, and achemotherapeutic agent. A peptide dosage may be about 1 to 10 mg perday, for example, when a patient is an adult. However, the dosage variesdepending on the age and weight of a patient, an administration method,and the like, and therefore, a person skilled in the art determines thedosage as appropriate.

The pharmaceutical composition of the present invention is not construedas being limited, but is thought to be useful for killing or the like ofcancer cells due to the following action mechanism. When thepharmaceutical composition of the present invention is administered to aspecific cancer patient, the peptide in the pharmaceutical compositionis presented on the surface of the antigen presenting cell in a state inwhich the peptide binds to an HLA molecule. The CTLs recognize thepeptide on such an antigen presenting cell and is thus activated,followed by proliferation and systemic circulation. When CTLs that arespecific to the peptide enter the cancer tissue, the CTLs recognize thesame peptide derived from the specific cancer antigen that naturallybinds to the HLA molecule present on the surface of the cancer cell, andthen kill the cancer cell. The pharmaceutical composition can contributeto treatment of cancer due to this action.

The pharmaceutical composition can be used for not only treatment ofcancer but also prevention of cancer. For example, when thepharmaceutical composition of the present invention is administered to ahealthy human body, CTLs are induced, and the induced CTLs remain in thebody. Therefore, when a specific cancer occurs, it is possible to injurethe cancer cells. Similarly, a recurrence of cancer may be prevented bythe administration to the human body that has undergone treatment ofcancer.

All cancers in which MUC1 is expressed are assumed as cancer to betreated or prevented. More specifically, examples of target cancersinclude pancreatic cancer, hepatocellular cancer, prostate cancer, lungcancer, breast cancer, bowel cancer, blood cancer, brain tumor, kidneycancer, and skin cancer, but are not construed as being limited. Forexample, MUC1 from which the peptide of the present invention is derivedis overexpressed in pancreatic cancer, and therefore, it is thought thatthe peptide of the present invention is effective in treatment orprevention of pancreatic cancer. When a plurality of types of cancers tobe treated or prevented are present, the pharmaceutical composition ofthe present invention can contain a plurality of types of activecomponents such as an immunogenic peptide.

The pharmaceutical composition of the present invention can beadministered to a patient in a form of a preparation of apharmaceutically acceptable salt obtained by dissolving thepharmaceutical composition in a water-soluble solvent. Examples of theform of such a pharmaceutically acceptable salt include forms of aphysiologically acceptable water-soluble salt such as a sodium salt, apotassium salt, a magnesium salt, or a calcium salt that is buffered ata physiological pH. A water-insoluble solvent can also be used inaddition to a water-soluble solvent, and examples of the water-insolublesolvent include alcohols such as ethanol and propylene glycol.

A preparation containing the pharmaceutical composition of thisembodiment can contain agents for various purposes, and examples of suchagents include a preservative and a buffer. Examples of the preservativeinclude sodium bisulfite, sodium bisulfate, sodium thiosulfatebenzalkonium chloride, chlorobutanol, thimerosal, phenylmercuricacetate, phenylmercuric nitrate, methylparaben, polyvinyl alcohol,phenylethyl alcohol, ammonia, dithiothreitol, and β-mercaptoethanol.Examples of the buffer include sodium carbonate, sodium borate, sodiumphosphate, sodium acetate, and sodium bicarbonate. These agents can bepresent in such an amount that the pH of a system can be maintainedbetween 2 and 9, and preferably between 4 and 8.

There is no particular limitation on the formulation of thepharmaceutical composition of the present invention, but examplesthereof include injections (an intramuscular injection, a hypodermicinjection, an intradermal injection), oral formulations, and nasalformulations when a form of vaccine is used. When the pharmaceuticalcomposition of the present invention is in a form of vaccine, a mixedcocktail vaccine containing a plurality of types of active componentsmay be used. For example, such vaccine can contain a plurality of typesof active components that is a combination of any two or more peptidesof Sequence ID Nos. 1 to 12 or other active components.

The vaccine of the present invention may be inert component-containingvaccine containing a component other than the pharmaceuticalcomposition, the component being inert itself and having an effect offurther improving the effect of the pharmaceutical composition asvaccine. Examples of the inert component include an adjuvant and atoxoid. Examples of the adjuvant include sedimentary adjuvants such asaluminum hydroxide, aluminum phosphate, and potassium phosphate, andoily adjuvants such as Freund's complete adjuvant and Freund'sincomplete adjuvant.

When the pharmaceutical composition of the present invention is presentin a form of vaccine, it is preferable to administer the pharmaceuticalcomposition of the present invention to the body by an injection orinfusion through intradermal administration, hypodermic administration,intravenous administration, intramuscular administration, or the like,or by transdermal inhalation or inhalation through a mucous membrane ofthe nose, the pharynx, or the like. A dose thereof can be set to bebetween such an amount that a cytotoxic T cell can be significantlyinduced and such an amount that a significant number of non-cancer cellsare not injured.

The pharmaceutical composition of the present invention is intended tobe not only administered to a human body but also used outside the body.More specifically, the pharmaceutical composition of the presentinvention may be used to stimulate an antigen presenting cell in vitroor ex vivo and increase a CTL-inducing activity. For example, thefollowing is description of an example of a case where thepharmaceutical composition of the present invention is used in adendritic cell therapy for cancer. The pharmaceutical composition of thepresent invention can be administered to a patient requiring treatmentor prevention of cancer by bringing the pharmaceutical composition intocontact with an antigen presenting cell such as a dendritic cell derivedfrom the patient in advance, and then putting the antigen presentingcell back to the body of the patient. The peptide contained in thepharmaceutical composition can be introduced into the antigen presentingcell using a lipofection method, an injection method, or the like. Whena polynucleotide coding for the peptide of the present invention is usedfor such an application, the polynucleotide can be introduced into theantigen presenting cell using a method known in the art. The antigenpresenting cell derived from a patient may be transformed in vitro, forexample, with the target polynucleotide or a vector coding for thepolynucleotide using a lipofection method, an electroporation method, amicroinjection method, a cell fusion method, a DEAE dextran method, acalcium phosphate method, or the like.

3. Immunity-Inducing Agent

An immunity-inducing agent according to the present invention contains,as an active component, a peptide that includes eight or moreconsecutive amino acid residues of one or more amino acid sequencesselected from the group consisting of Sequence ID Nos. 1 to 12, and thatconsists of eleven or less amino acids, preferably ten or less aminoacids, and more preferably nine or less amino acids, in total, forexample. The peptide contained in the immunity-inducing agent may alsohave the amino acid sequence of one of Sequence ID Nos. 1 to 12. Thepeptide is as defined above.

It is thought that the peptide of the present invention is presented onan antigen presenting cell and thus induces immunity. Therefore, theactive component of the immunity-inducing agent of the present inventionis not limited to the peptide of the present invention, and may be acomponent that can induce immunity directly or indirectly. For example,a polynucleotide coding for the peptide of the present invention or avector including the polynucleotide, an antigen presenting cell thatpresents a complex between the peptide and an HLA molecule on itssurface, or an exosome secreted by the antigen presenting cell, orcombinations thereof may be used. Examples of the antigen presentingcell to be used include a macrophage and a dendritic cell, and it ispreferable to use a dendritic cell, which has a high ability to induceCTLs.

The immunity-inducing agent of the present invention is intended to benot only administered to a human body but also used outside the body.More specifically, the immunity-inducing agent of the present inventionmay be used to stimulate an antigen presenting cell in vitro or ex vivoand increase a CTL-inducing activity. For example, the following isdescription of an example of a case where the immunity-inducing agent ofthe present invention is used in a dendritic cell therapy. Theimmunity-inducing agent of the present invention can be administered toa patient requiring immunity induction by bringing the immunity-inducingagent into contact with an antigen presenting cell such as a dendriticcell derived from the patient in advance, and then putting the antigenpresenting cell back to the body of the patient. The peptide containedin the immunity-inducing agent can be introduced into the antigenpresenting cell using transfection via a liposome (lipofection method),an injection method, or the like. When a polynucleotide coding for thepeptide of the present invention is used for such an application, thepolynucleotide can be introduced into the antigen presenting cell usinga method known in the art. The antigen presenting cell derived from apatient may be transformed in vitro, for example, with the targetpolynucleotide or a vector expressing the polynucleotide using alipofection method, an electroporation method, a microinjection method,a cell fusion method, a DEAE dextran method, a calcium phosphate method,or the like.

The “immunity induction” as used herein means that an immune reaction isinduced, and a CTL-inducing activity of the antigen presenting cell aswell as a cytotoxic activity of CTLs against cancer cells, for example,are thus increased. The “CTL induction” as used herein means that, invitro or in vivo, CTLs that specifically recognize a certain antigen areinduced or proliferated, or naive T cells are differentiated intoeffector cells that has an ability (cytotoxic activity) to kill targetcells such as cancer cells, and/or the cytotoxic activity of CTLs isincreased, due to the peptide of the present invention being presentedon the surface of an antigen presenting cell. The CTL-inducing activitycan be measured by evaluating the production of a cytokine (e.g.,interferon (IFN)-γ) by CTLs. For example, the CTL-inducing activity maybe measured by evaluating, using a known highly sensitive immunoassaysuch as ELISPOT (Enzyme-Linked ImmunoSpot) or ELISA (Enzyme-LinkedImmunoSorbent Assay), an increase in the number of cytokine-producingcells that have been induced from precursor cells by an antigenpresenting cell such as a peripheral blood mononuclear cell stimulatedby the peptide of the present invention. The cytotoxic activity can alsobe measured using a known method such as a ⁵¹Cr releasing method. Whenthe above-mentioned activities increase significantly, for example, by5% or more, 10% or more, or 20% or more, or preferably 50% or more,compared with control samples, immunity and CTLs can be evaluated asbeing induced.

4. Method for Manufacturing Antigen Presenting Cell

A method for manufacturing an antigen presenting cell according to thepresent invention includes a step of bringing a peptide that includeseight or more consecutive amino acid residues of one or more amino acidsequences selected from the group consisting of Sequence ID Nos. 1 to12, and that consists of eleven or less amino acids, preferably ten orless amino acids, and more preferably nine or less amino acids, in totalinto contact with an antigen presenting cell in vitro, for example. Thepeptide used in the manufacturing method of the present invention mayalso have the amino acid sequence of one of Sequence ID Nos. 1 to 12.The peptide is as defined above.

It is thought that the peptide used in the manufacturing method of thepresent invention binds to an HLA class-I molecule on the surface of anantigen presenting cell and is presented to CTLs as an antigen peptide,and the CTL activity of the antigen presenting cell is thus induced.Therefore, a component to be brought into contact with an antigenpresenting cell is not limited to the peptide of the present invention,and may be a component that can induce CTLs directly or indirectly. Forexample, a polynucleotide coding for the peptide or a vector includingthe polynucleotide, an antigen presenting cell that presents a complexbetween the peptide and an HLA molecule on its surface, or an exosomesecreted by the antigen presenting cell, or combinations thereof may beused. Examples of the antigen presenting cell to be used include amacrophage and a dendritic cell, and it is preferable to use a dendriticcell, which has a high ability to induce CTLs.

The antigen presenting cell manufactured using the manufacturing methodof the present invention is intended to be used not only as an activecomponent of the above-mentioned pharmaceutical composition orimmunity-inducing agent but also in an immunotherapy or the like. Forexample, the following is description of an example of a case where themanufactured antigen presenting cell is used in a dendritic cell therapyfor cancer. The manufactured antigen presenting cell can be administeredto a patient requiring immunity induction by bringing the manufacturedantigen presenting cell into contact with an antigen presenting cellsuch as a dendritic cell that are derived from the patient and has a lowability to induce CTLs in advance, and then putting the manufacturedantigen presenting cell back to the body of the patient. The peptide ofthe present invention can be introduced into the antigen presenting cellusing transfection via a liposome (lipofection method), an injectionmethod, or the like. When a polynucleotide coding for the peptide of thepresent invention is used for such an application, the polynucleotidecan be introduced into the antigen presenting cell using a method knownin the art. The antigen presenting cell derived from a patient may betransformed in vitro, for example, with the target polynucleotide or avector coding for the polynucleotide using a lipofection method, anelectroporation method, a microinjection method, a cell fusion method, aDEAE dextran method, a calcium phosphate method, or the like.

Example 1

Hereinafter, the present invention will be described more specificallyby use of examples, but the present invention is not limited to theexamples.

Specifically, procedures of prediction, experiment, and evaluation inthese examples were carried out based on the active learning experimentdesign described in WO 2006/004182, and rules were constructed byrepeating the following steps as a whole.

(1) A trial of a lower-order learning algorithm, which will be describedlater, was carried out once. That is, a plurality of hypotheses weregenerated by random resampling from accumulated data, and with regard torandomly expressed candidate query points (peptides), a point thatshowed the largest distribution of predicted values was selected as aquery point to be subjected to an experiment.

(2) The peptide at the selected query point was prepared by a synthesisand purification method, which will be described later, and the actualbinding ability was measured by an experiment, which will be describedlater, and added to the accumulated data.

In accordance with such an active learning method, the number ofrepetitions of the binding experiment for peptides consisting of 9 aminoacid residues, which would otherwise have to be carried out for the 500billion (=20⁹) or more combinations of all the candidates forHLA-binding peptides, could be reduced.

Based on the rules as described above, the amino acid sequences ofSequence ID Nos. 1 to 12 were extracted.

Synthesis and Purification of Peptide

Peptides having amino acid sequences of Sequence ID Nos. 1 to 12 weremanually synthesized with the Merrifield solid-phase method using Fmocamino acids. After deprotection, reverse phase HPLC purification wascarried out using a C18 column to give a purity of 95% or higher.MALDI-TOF mass spectrometry (AB SCIEX MALDI-TOF/TOF5800) was carried outto identify the peptides and confirm their purities. The peptides werequantified with Micro BCA assay (Thermo Scientific) using BSA as astandard protein.

Experiment of Binding Peptide to HLA-A*24:02 Molecule

The ability of each of the peptides to bind to an HLA-A*24:02 molecule,which is an HLA-A*24:02 gene product, was measured using C1R-A24 cellsexpressing the HLA-A*24:02 molecule (cells prepared by ProfessorMasafumi TAKIGUCHI, Kumamoto University being supplied with permissionby Assistant Professor Masaki YASUKAWA, Ehime University).

First, C1R-A24 cells were exposed to acidic conditions at a pH of 3.3for 30 seconds, and thus an endogenous peptide that originally binds tothe HLA-A*24:02 molecule, and a light chain β2m, which is associatedwith HLA class-I molecules in common, were dissociated and removed.After neutralization, a purified β2m was added to C1R-A24 cells, and theobtained product was added to a series of dilutions of the peptide andincubated on ice for 4 hours. Staining was carried out using afluorescently labeled monoclonal antibody 17A12, which recognizesassociation (MHC-pep) of the three members, that is, a HLA-A*24:02molecule, a peptide, and β2m, which had reassociated during theincubation.

Thereafter, the MHC-pep count per C1R-A24 cell (proportional to theintensity of fluorescence of the above-mentioned fluorescent antibody)was quantitatively measured using a fluorescence-activated cell sorterFACScan (Becton-Dickinson). A binding-dissociation constant Kd valuebetween the HLA-A*24:02 molecule and the peptide was calculated from theaverage intensity of fluorescence per cell using a method that wasreported in a paper (Udaka et al., Immunogenetics, 51, 816-828, 2000) bythe inventors of the present invention.

Experiment of Binding Peptide to HLA-A*02:01 Molecule

The ability of each of the peptides to bind to an HLA-A*02:01 molecule,which is an HLA-A*02:01 gene product, was measured using a cell line T2(purchased from ATCC) expressing the HLA-A*02:01 molecule.

The T2 cells and purified β2m were added to a series of serial dilutionsof the peptide whose binding ability would be measured, and thenincubation was carried out at 37° C. for 4 hours. The HLA-A*02:01molecule that had increased in an expression amount in apeptide-concentration-dependent manner up to this point was stainedusing a fluorescently labeled monoclonal antibody BB7.2, which isspecific to an association type.

Thereafter, the amount of fluorescence per cell was measured using aflow cytometer, and a dissociation constant Kd value was calculatedusing a method that was reported in a paper (Udaka et al.,Immunogenetics, 51, 816-828, 2000) by the inventors of the presentinvention.

Experiment of Binding Peptide to HLA-A*02:06 Molecule

The ability of each of the peptides to bind to an HLA-A*02:06 molecule,which is an HLA-A*02:06 gene product, was measured using RA2.6 cells(cell line that had been newly produced in Kochi University) produced byintroducing the cDNA of the HLA-A*02:06 gene into a mouse TAP(transporter associated with antigen processing)-deficient cell lineRMAS.

First, RA2.6 cells were cultured at 26° C. overnight. After HLA-A*02:06molecules to which the peptide did not bind accumulated on the cellsurface, a series of dilutions of the peptide were added thereto, andbinding was carried out at 26° C. for 60 minutes.

Thereafter, the cells were cultured at 35° C. for 4 hours. As a result,blank HLA-A*02:06 molecules to which the peptide did not bind weredenatured, and thus their conformation was lost. A fluorescently labeledmonoclonal antibody BB7.2, which specifically recognizes apeptide-binding HLA-A*02:06 molecule, was added thereto, incubation wascarried out on ice for 20 minutes, and thus the cells were stained.

Thereafter, the amount of fluorescence per cell was measured using aflow cytometer, and a dissociation constant Kd value was calculatedusing a method that was reported in a paper (Udaka et al.,Immunogenetics, 51, 816-828, 2000) by the inventors of the presentinvention.

Evaluation Results of Binding Experiments

As a result, as shown in a table below, the data from the experiment ofbinding the peptides of the present invention to the HLA molecules wereobtained.

TABLE 2 Amino acid sequence Location in Data of binding experiment(Sequence ID No.) MUC1 to A*24:02 to A*02:01 to A*02:06FLGLSNIKF (Seq. ID No. 1) 1086 −7.073261692 −5.390853526 −4.386007431SVPVTRPAL (Seq. ID No. 2)  100 −7.241740934 −3.903379214 −5.627441171GVPGWGIAL (Seq. ID No. 3) 1155 −5.927200496 −4.54796761 −5.763029942AFREGTINV (Seq. ID No. 4) 1106 −5.834149762 >−3 >−3AASRYNLTI (Seq. ID No. 5) 1128 −6.148024331 −4.811130083 −3.824201259LQRDISEMF (Seq. ID No. 6) 1069 −6.646212952 >−3 −4.260522119HHSDTPTTL (Seq. ID No. 7)  997 −5.867440069 >−3 >−3SFFFLSFHI (Seq. ID No. 8) 1041 −7.065109329 >−4 −3.980495237TLAFREGTI (Seq. ID No. 9) 1104 −5.973982835 >−3  −3.08622966STGVSFFFL (Seq. ID No. 10) 1037 −5.171414811 −5.121912366 −5.208079724GQDVTSVPV (Seq. ID No. 11)   95 >−3 −6.437064471 −6.349002291FSAQSGAGV (Seq. ID No. 12) 1148 >−3 −4.74018 −5.661074048

It should be noted that the amino acid sequences of Sequence ID Nos. 1to 12 are derived from the entire sequence (Sequence ID No. 13)(>sp|P15941|MUC1_HUMAN Mucin-1 OS=Homo sapiens GN=MUC1 PE=1 SV=3) of apredetermined MUC1 genome protein registered in GENBANK.

Peptide Immunity Induction Testing

(1) Preparation of Peptide-Stimulated Dendritic Cell

Day 0 to 9 (Induction of Dendritic Cell)

Of the peripheral blood monocytes obtained through pheresis from apatient (0) that had undergone a dendritic cell/CTL therapy againstMUC1, a cell fraction that adhered to a culture flask was cultured in anAIM-CM culture medium (manufactured by Gibco) at 37° C. for 10 days.During the culture, 15 μl of IL-4 and 30 μl of a granulocyte monocytecolony-stimulating factor (GM-CSF) added to the culture medium on Day 0and Day 3, and 15 μl of IL-4, 30 μl of GM-CSF, and 75 μl of a tumornecrosis factor (TFN)-α were added thereto on Day 5.

Day 10 (Peptide Stimulation and Dendritic Cell Collection)

Induced dendritic cells were collected in a new AIM-CM culture medium,and the peptide of the present invention (Sequence ID Nos. 1 to 12) wasadded thereto to a concentration of 20 μg/ml. Thereafter, the culturemedium containing the dendritic cells was cultured at 37° C. for 2hours. The following peptides were used as positive controls andnegative controls.

Positive control for HLA-A*24:02(EBV LMP2, 419-427: TYGPVFMCL (Sequence ID No. 14))Negative control for HLA-A*24:02(HIV env gp160, 584-592: RYLRDQQLL (Sequence ID No. 15))Positive control for HLA-A*02:01(Flu A MP, 58-66: GILGFVFTL (Sequence ID No. 16))Negative control for HLA-A*02:01(HIV gap p17, 77-85: SLYNTVATL (Sequence ID No. 17))Positive control for HLA-A*02:06(EBV LMP2 453-461: LTAGFLIFL (Sequence ID No. 18))Negative conrol for HLA-A*02:06(HIV gap p24 341-349: ATLEEMMTA (Sequence ID No. 19))

The dendritic cells were collected and washed three or more times usinga sufficient amount of an AIM-CM culture medium, and then the number ofcells was counted.

(2) Preparation of CD8T Cells

Day 0 to 9

Of the peripheral blood monocytes obtained through pheresis from apatient that had undergone treatment using the above-mentioned vaccinetwice or more, a floating cell fraction (containing lymphocytes) thatdid not adhere to a culture flask was cultured in an AIM-CM culturemedium (manufactured by Gibco) at 37° C. for 10 days. During theculture, 40 μl of IL-2 was added to the culture medium on Day 4 and Day6.

Day 10

CD8T cells were isolated from the culture medium using a CD8 negativeselection kit (manufactured by Miltenyi), and the number of cells wascounted.

(3) Coculture

The dendritic cells and CD8T cells obtained in the above (1) and (2)were cocultured in an AIM-CM culture medium at 37° C. in the followingconditions.

-   -   CD8T cell: 5×10⁵ cells/well    -   Dendritic cell: 2×10⁵ cells/well

Day 12 or 13

To the above-mentioned culture medium, 0.4 ml/well of an AIM-CM culturemedium containing 20 U/ml of IL-2 was added.

(5) ELISA Assay

Day 17

The culture supernatant of the coculture of the T cells and thedendritic cells pulsed with the above-mentioned peptides on Day 7 wasdiluted to four steps, that is, ×1, ×5, ×25, and ×125, and the dilutionstep within a measurement limit was identified using Human IFN-γ ELISAMAX Deluxe Set (manufactured by BioLegend). Thereafter, at theidentified dilution step, each sample was measured three times. FIGS. 1to 4 respectively show typical results of ELISA assay from a patientwith an HLA type of 24:02/33:03, a patient with an HLA type of24:02/33:03, a patient with an HLA type of 11:01/24:02, a patient withan HLA type of 02:06/24:02, and a patient having an HLA type of02:01/02:06.

The present invention has been described based on the examples. Theexamples are merely exemplary, and a person skilled in the art wouldunderstand that various modified examples are possible, and suchmodified examples are also within a scope of the present invention.

1. A peptide comprising eight or more consecutive amino acid residues ofamino acid sequence of one of Sequence ID Nos. 1 to 12, and consistingof eleven or less amino acid residues.
 2. The peptide according to claim1, wherein one or several amino acids in the amino acid sequence aresubstituted, inserted, deleted, or added, and the peptide hasimmunogenicity.
 3. The peptide according to claim 2, wherein an aminoacid at a 2-position of the amino acid sequence is substituted withtyrosine, phenylalanine, methionine, tryptophan, valine, leucine orglutamine, and/or a C-terminal amino acid is substituted withphenylalanine, leucine, isoleucine, tryptophan, methionine, or valine.4. A pharmaceutical composition for treatment or prevention of cancer,comprising the peptide according to claim
 1. 5. The pharmaceuticalcomposition according to claim 4, which is in a form of vaccine.
 6. Thepharmaceutical composition according to claim 4, wherein the peptide canbind to one or more types of HLA molecules.
 7. An immunity-inducingagent comprising the peptide according to claim
 1. 8. Theimmunity-inducing agent according to claim 7, which is used to induce acytotoxic T cell.
 9. The immunity-inducing agent according to claim 7,wherein the peptide can bind to one or more types of HLA molecules. 10.A method for manufacturing an antigen presenting cell having aCTL-inducing activity, the method comprising a step of bringing thepeptide according to claim 1 into contact with an antigen presentingcell in vitro.